U.S. patent application number 10/365242 was filed with the patent office on 2003-12-18 for photoelectrochemical molecular comb.
Invention is credited to Brown, Gilbert M., Ferrell, Thomas L., Thundat, Thomas G..
Application Number | 20030232426 10/365242 |
Document ID | / |
Family ID | 27732693 |
Filed Date | 2003-12-18 |
United States Patent
Application |
20030232426 |
Kind Code |
A1 |
Thundat, Thomas G. ; et
al. |
December 18, 2003 |
Photoelectrochemical molecular comb
Abstract
A method, system, and apparatus are provided for separating
molecules, such as biomolecules. The method, system, and apparatus
utilize an electrochemical cell having at least to electrodes, one
electrode comprising a photo-sensitive material capable of
generating a photopotential. Molecules are moved through an
electrolyte medium between the at least two electrodes based upon
localized photopotentials.
Inventors: |
Thundat, Thomas G.;
(Knoxville, TN) ; Ferrell, Thomas L.; (Knoxville,
TN) ; Brown, Gilbert M.; (Knoxville, TN) |
Correspondence
Address: |
Daniel G. Radler, Esq.
c/o Quarles & Brady LLP
411 E. Wisconsin Avenue
Milwaukee
WI
53202
US
|
Family ID: |
27732693 |
Appl. No.: |
10/365242 |
Filed: |
February 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10365242 |
Feb 12, 2003 |
|
|
|
10077633 |
Feb 15, 2002 |
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Current U.S.
Class: |
435/287.2 ;
205/777.5 |
Current CPC
Class: |
G01N 27/305
20130101 |
Class at
Publication: |
435/287.2 ;
205/777.5 |
International
Class: |
C12M 001/34; G01F
001/64; G01N 027/26; G01N 033/50 |
Goverment Interests
[0002] The invention was made with government support under
contract DE-AC05-00OR22725, awarded by the United States Department
of Energy to UT-Battelle, LLC, the United States Government has
certain rights in this invention.
Claims
What is claimed is:
1. An apparatus, comprising: a photo-sensitive electrode; an
electrolyte medium in contact with the photo-sensitive electrode; a
counter electrode; a voltage source electrically coupled to the
photo-sensitive electrode and the counter electrode, wherein a
voltage generates a depletion region in the photo-sensitive
electrode; and at least one photon energy source incident upon the
depletion region, wherein the photon energy source generates photon
energy such that photon energy contacting the depletion region
forms electron-hole pairs, wherein either or both of the photon
energy source and/or photo-sensitive electrode is movable relative
to the other.
2. The apparatus of claim 1, wherein the photo-sensitive electrode
is a semi-conductive material.
3. The apparatus of claim 2, wherein the semi-conductive material
is selected from the group consisting of Si, Ge, GaAs, TiO.sub.2,
CdS, and ZnO.
4. The apparatus of claim 1, wherein the electrolyte medium is a
film.
5. The apparatus of claim 4, wherein the film comprises a
composition selected from the group consisting of polyacrylamide,
agarose, polymethyl methacrylate, and dextran.
6. The apparatus of claim 1, wherein the counter electrode
comprises a conductive organic material.
7. The apparatus of claim 1, wherein the counter electrode
comprises a conductive inorganic material.
8. The apparatus of claim 1, wherein the counter electrode
comprises a mixed inorganic/organic conductor.
9. The apparatus of claim 1, wherein the photon energy source
produces a beam of light to create a localized photopotential in
the electrolyte medium.
10. The apparatus of claim 1, wherein the photon energy source
produces a beam of a desired geometry.
11. The apparatus of claim 1, wherein the photon energy source is
intermittent.
12. The apparatus of claim 1, wherein the photon energy source is
moved relative to the photo-sensitive electrode.
13. The apparatus of claim 1, wherein the voltage source comprises
a potentiostat.
14. The apparatus of claim 1, wherein the voltage is modulated.
15. The apparatus of claim 1, wherein the surface of the
photo-sensitive electrode is artificially patterned.
16. The apparatus of claim 1, wherein the voltage is alternated to
generate an alternating pulsed potential between the electrode.
17. The apparatus of claim 6, 7, or 8, wherein the counter
electrode is optically transmissive and electrically
conductive.
18. The apparatus of claim 1, wherein the counter electrode is
substantially planar and parallel to the photo-sensitive
electrode.
19. A method for separating molecules, comprising applying a
voltage to a photo-sensitive electrode and a counter electrode to
generate a depletion region on the photo-sensitive electrode,
wherein the electrodes are separated by an electrolyte medium in
contact with the photo-sensitive electrode, wherein the electrolyte
medium comprises a plurality of analytes; contacting the depletion
region with a photon energy source, wherein the photon energy
source generates photon energy such that photon energy contacting
the depletion region forms electron-hole pairs that are separated
by the potential to form a photopotential; and continually changing
the location of contact of the photon energy with the depletion
region parallel to the photo-sensitive electrode such that a
photopotential is propagated across the photo-sensitive electrode
and proximal to the location of the analytes thereby causing the
analytes to migrate relative to the photopotential.
20. The method of claim 19, wherein the analytes comprise
biomolecules.
21. The method of claim 20, wherein the biomolecules are selected
from the group consisting of polynucleotides, oligonucleotides,
proteins, polypeptides, and peptides.
22. The method of claim 19, wherein the photon energy is emitted by
a laser.
23. The method of claim 19, wherein the photo-sensitive electrode
is a semi-conductive material.
24. The method of claim 23, wherein the semi-conductive material is
selected from the group consisting of Si, Ge, GaAs, TiO.sub.2, CdS,
and ZnO.
25. The method of claim 19, wherein the electrolyte medium is a
film.
26. The method of claim 25, wherein the film comprises a
composition selected from the group consisting of polyacrylamide,
agarose, polymethyl methacrylate, and dextran.
27. The method of claim 19, wherein the counter electrode comprises
a conductive organic material.
28. The method of claim 19, wherein the counter electrode comprises
a conductive inorganic material.
29. The method of claim 19, wherein the counter electrode comprises
a mixed inorganic/organic conductor.
30. The method of claim 19, wherein the photon energy source
produces a beam of light to create a localized photopotential in
the tonically conductive medium.
31. The method of claim 19, wherein the photon energy source
produces a beam of a desired geometry.
32. The method of claim 19, wherein the photon energy source is
intermittent.
33. The method of claim 19, wherein the photon energy source is
moved relative to the. photo-sensitive electrode.
34. The method of claim 19, wherein the voltage is applied between
the counter electrode and the substrate using a potentiostat.
35. The method of claim 19, wherein the voltage is modulated.
36. The method of claim 19, wherein the surface of the
photo-sensitive electrode is artificially patterned.
37. The method of claim 19, wherein the voltage is alternated to
generate an alternating pulsed photopotential.
38. The method of claim 27, 28, or 29, wherein the counter
electrode is optically transmissive and electrically
conductive.
39. The method of claim 20, wherein the biomolecule comprises a
detectable label.
40. The method of claim 39, wherein the detectable label is
selected from the group consisting of an radioisotope, a dye, a
fluorescent molecule, a luminescent molecule, and an enzyme.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. application Ser.
No. 10/077,633, filed Feb. 15, 2002, the disclosure of which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0003] The invention generally relates to the molecular
characterization of molecules. Specifically, the invention relates
to fractionation of heterogeneous mixtures of particles or
molecules on the basis of characteristics such as size and/or
charge and also the analysis of electrochemical properties of
particles.
BACKGROUND
[0004] Analysis of a sample of biological origin frequently
requires the separation of mixtures of biomolecules, such as
nucleic acids, proteins, and polypeptides, which often have limited
sample size. Electrophoresis, in which charged molecules move in a
liquid under the influence of an electric field, has long been the
method of choice for separating many classes of biomolecules. This
method takes of advantage of differing migration velocities,
v.sub.ep, of different molecules.
[0005] The migration velocity, v.sub.ep, is the distance (L) a
molecule or particle moves per unit time (t). The migration
velocity is the product of the electrophoretic mobility,
.mu..sub.ep, multiplied by the electric field strength E (units of
volts/cm):
v.sub.ep=.mu..sub.ep.times.E.mu..sub.ep=q/6.pi..eta.R
[0006] where q is the charge on the particle and .eta. is the
viscosity of the medium and R is the radius of the particle. The
velocity is, thus, directly proportional to the charge on the
particle and the field strength and inversely proportional to the
size of the particle and the viscosity of the medium. For
relatively large particles or biomolecules, the charge increases as
the size of the molecule increases, and the charge to mass (or
radius) ratio becomes nearly constant. Under these circumstances if
the electrophoresis is carried out in the presence of a gel
composed of agarose (agarose gel electrophoresis) or crosslinked
polyacrylamide (polyacrylamide gel electrophoresis), the gel
structure creates a molecular sieving effect that allows the
molecules or particles to be separated on the basis of size.
[0007] Capillary gel electrophoresis is typically carried out in 50
.mu.m diameter capillaries that are 10 cm to 1 m long with a field
strength that is generally in the range of 100 V/cm to 500 V/cm,
and requires a high-applied voltage greater, typically greater than
1 KV. Heat generation is directly proportional to the square of the
applied voltage, and the voltages required to achieve separation in
capillary electrophoresis may cause degradation of sensitive
samples.
[0008] A subject of a considerable amount of research in recent
years has been microscale fluid handling systems that perform fast,
automated, high-resolution sample preparation, reaction, and
separation. Currently, this is being accomplished through advances
in microfluidics. The idea is that once the. manipulation of fluids
can be mastered on the microscale, key experiments for biomolecule
separation and analysis can be integrated and automated--all on a
mass-produced chip. In microfluidic-based devices, nucleic acid
molecules, proteins, polypeptides and other such molecules are
transported, manipulated, and separated through miniature channels
embedded into the chip. Detection systems can also be integrated
into the chip or affixed externally as a separate component for
seamless, automated and highly sensitive detection.
SUMMARY
[0009] The invention provides an apparatus, comprising a
photo-sensitive electrode; an electrolyte medium in contact with
the photo-sensitive electrode; a counter electrode; and a voltage
source. The voltage source is electrically coupled to the
photo-sensitive electrode and the counter electrode such that when
a voltage is applied to the electrodes a depletion region is
generated in the photo-sensitive electrode. At least one photon
energy source incident upon the depletion region, wherein the
photon energy source generates photon energy such that photon
energy contacting the depletion region forms electron-hole pairs,
wherein either or both of the photon energy source and/or
photo-sensitive electrode is movable relative to the other. In one
aspect of the invention the photo-sensitive electrode is a
semi-conductive material. The semiconductive electrode may be
selected from the group consisting of Si, Ge, GaAs, TiO2, CdS, and
ZnO. In another aspect of the invention the photon energy source
produces a beam of light to create a localized photopotential in
the electrolyte medium. In yet another aspect of the invention the
photon energy source is moved relative to the photo-sensitive
electrode. The voltage applied to the electrodes may be reversed or
alternated in order to reverse the electric field between the
electrodes intermittently. In one aspect of the invention, the
counter electrode is optically transmissive and electrically
conductive.
[0010] The invention also provides a method for separating
molecules. The method comprises applying a voltage to a
photo-sensitive electrode and a counter electrode to generate a
depletion region on the photo-sensitive electrode, wherein the
electrodes are separated by an electrolyte medium in contact with
the photo-sensitive electrode, wherein the electrolyte medium
comprises a plurality of analytes. The depletion region is then
contacted with a photon energy source, wherein the photon energy
source generates photon energy such that photon energy contacting
the depletion region forms electron-hole pairs that are separated
by the potential to form a photopotential. The photon energy source
is moved relative to the semiconductive surface such that a
photopotential is propagated across the photo-sensitive electrode
and proximal to the location of the analytes thereby causing the
analytes to migrate relative to the photopotential. In one aspect
of the invention the analytes comprise biomolecules such as
polynucleotides, oligonucleotides, proteins, polypeptides, and/or
peptides. The molecules may be detectably labeled with agents known
in the art including, for example, a radioisotope, a dye, a
fluorescent molecule, a luminescent molecule, and/or an enzyme.
[0011] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0012] FIG. 1 is a schematic of an apparatus of the invention.
[0013] FIG. 2 collectively shows an electrochemical cell of the
apparatus of FIG. 1.
[0014] FIG. 3 shows an energy diagram at the interface between a
photo-sensitive electrode and an electrolyte medium during
operation of the apparatus of FIG. 1. Ecb is the conduction band;
Evb is the valence band and Ef is the Fermi level.
[0015] FIG. 4 is a diagram showing the modified surface of a
photo-sensitive electrode of the invention useful in separating
molecules.
[0016] FIG. 5 is a diagram showing the movement of a biomolecule
within the electrolyte solution as the potential is alternated
between the electrodes.
[0017] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0018] The invention provides an apparatus, system and method for
manipulating charged biomolecules using photon energy. The
invention utilizes electrical potentials and photopotentials to
move biomolecules on or within an electrolyte medium (e.g., an
ionically conductive medium).
[0019] The invention provides a microscale method for performing
chemical separation of biomolecules such as polynucleotides,
oligonucleotides, proteins, polypeptides, and peptides on a chip
using photoelectrochemistry. Advantageously, the invention provides
a method of separation and analysis that requires neither miniature
channels nor capillary tubes for the separation and analysis of the
same class or complex mixtures of biomolecules. However, the
apparatus, system, and methods of the invention may be combined
with further analytical systems including microfluidic chips known
in the art. The combination of the methods and systems of the
invention are used for a first degree of separation and further
analytical processes are then used for further characterization, if
needed.
[0020] The photoelectrochemical molecular comb technology disclosed
herein utilizes electrokinetics and semiconductor
photoelectrochemistry to create a spatially confined electric field
on a microchip, which is used to transport and separate
biomolecules without the use of channels. This innovative
technology has the potential to provide substantial cost and
performance advantages for users, including minimizing the amount
of the sample required, minimizing the time for processing,
increasing analysis flexibility and complexity, and improving
separation resolution.
[0021] The invention provides a general light-directed method for
separating molecules. This objective is accomplished by creating a
localized photovoltage that moves analyte molecules, as opposed to
creating a static field gradient. The methods and apparatus of the
invention provide an advantageous and compact system for the
separation of biomolecules and particles.
[0022] Generally, a pair of electrodes (anode and cathode) are
maintained in contact with an electrolyte medium by, for example,
direct immersion in the electrolyte medium. The electrodes are
immersed in the electrolyte medium to allow a desired voltage
gradient to be maintained across, for example, a porous substrate
or gel when a direct current power source is attached to the
electrodes.
[0023] The external electric field applied between the electrodes
causes positively charged cations to migrate toward the negatively
charged electrode, or cathode, and negatively charged anions to
migrate toward the positively charged anode. In this manner,
analytes move toward the anode or cathode under the influence of
the electric field. In the absence of any other influences, the
analytes will travel through the electrolyte medium as discrete
zones, or bands, based on differences in solute mobility.
[0024] In the simplest mode of electrophoresis, free solution or
zone electrophoresis, a buffer solution consists solely of an
electrolyte medium. Analytes are separated purely on differences in
the electrophoretic velocity of analytes due to their
charge-to-size ratios.
[0025] By modulating electrical and photopotential between
electrodes, biomolecules such as proteins and/or polynucleotides
either seek out, or avoid, regions of high ionic current, depending
upon their charge characteristics. Thus, by changing the location
of the high-ionic current on a semiconductive electrode surface by
utilizing photon energy, control over the movement of charged
biomolecules can be accomplished. The spatial and temporal
modulation of potential between the electrodes (e.g., a
semiconductive electrode and counter electrode) through the use of
external photon energy (e.g., light) provides the basis to control
the electrokinetic forces and the movement of such charged
biomolecules as polypeptides, oligonucleotides, and
polynucleotides.
[0026] A biomolecule includes any number of various molecules. For
example a molecule or analyte of interest may be a nucleic acid
(e.g., DNA or RNA), a polypeptide (e.g., an antibody, protein,
enzyme), a biochemical (e.g., a lipid, hormone, fatty acids,
carbohydrate), pharmaceuticals, a chemical such as organics
including, for example, alkanes, alkenes, alkynes, dienes,
alicyclic hydrocarbons, arenes, alcohols, ethers, ketones,
aldehydes, cyclic hydrocarbons, carbonyls, carbanions, polynuclear
aromatics and derivatives of such organics, e.g., halide
derivatives, so long as the molecule has an overall net positive or
negative charge.
[0027] To understand the principle of this methodology, it will be
helpful to briefly review photoelectric properties of
semiconductors, or more specifically, those of an ES structure
formed by an electrolyte solution (E); and a semiconductor (S). The
photoelectric characteristics of this structure are closely related
to those of a standard Metal-Semiconductor (MS) device, which is
described in S. M. Sze, "The Physics of Semiconductors", 2nd
Edition, Chapt. 7 (Wiley Interscience 1981), the contents of which
are incorporated herein by reference.
[0028] Space-charge regions of small but finite thickness form at a
semiconductive/electrolyte interface in the presence of a bias
potential. In the case of the ES structure, an effective bias, in
the form of a junction potential, is present under all but very
special conditions. A space-charge region forms in response to the
distance dependence of charge density on the semiconductor. This
produces an electric potential gradient that changes the energy of
the semiconductor's valence and conduction bands ("band bending")
in the vicinity of the interface. This condition in turn reflects
the fact that, while there is a bias potential across the
interface, there is no net charge transfer at the interface when
equilibrium is reached.
[0029] In the presence of a reverse bias, the valence and
conduction band edges of an n-doped semiconductor bend upward near
the interface and electrons flow out of the interfacial region in
response to the corresponding potential gradient. As a result, a
majority carrier depletion layer is formed in the vicinity of the
interface. Light absorption in the semiconductor provides a
mechanism to create electron-hole pairs within this depletion
layer/region. The electron-hole pairs are split by the locally
acting electric field, and a corresponding photocurrent flows. It
is this latter effect that allows for the movement of charged
biomolecules in the electrolyte medium.
[0030] Band bending can be achieved by suitably polarizing the
semiconductor with respect to the electrolyte medium using a power
supply. The back contact to the semiconductor electrode is Ohmic in
character while the semiconductor-electrolyte medium interface acts
as a Schottky barrier. Therefore, most of the applied voltage is
dropped at the semiconductor-electrolyte medium interface creating
a space charge (depletion or accumulation) layer in the
semiconductor. The formation of a depletion or accumulation layer
depends on the bias and the type of semiconductor (i.e., n-type or
p-type). The nature of band bending can be changed from depletion
to accumulation by changing the sign of the applied voltage with
respect to the flat band potential of the semiconductor-electrolyte
interface.
[0031] Proper biasing of the semiconductor-electrolyte interface
results in the subsequent creation of a charge-depletion layer in
the semiconductor. Irradiation of the semiconductor-electrolyte
medium interface with photons of appropriate energy produces
electron-hole pairs in the depletion or accumulation layer. The
electric field in the depletion layer separates the electron-hole
pairs. For example, for an n-type semiconductor, the bands are-bent
upwards for a depletion layer, and therefore electron vacancies
(i.e., holes) come to the semiconductor-electrolyte interface
during illumination. In the case of an accumulation layer
irradiation causes the electrons to accumulate weakly at the
solid-electrolyte interface. Irradiation of the spot containing the
biomolecules with a focused beam of photons of energy greater than
the band gap of the photo-sensitive semiconductive electrode
generates new charge carriers in the charge-depletion layer. The
thereby-separated charge carriers reach the interface of the
semiconductor and electrolyte and create a localized
photovoltage.
[0032] The depletion layer present on the semiconductor exhibits
electrical characteristics similar to those of a capacitor with a
voltage-dependent capacitance. Illumination serves to lower the
impedance of the depletion layer.
[0033] The ES structure can also comprise an oxide layer between
the semiconductor and the electrolyte medium. The oxide layer will
pass current only above a characteristic ("threshold")
frequency.
[0034] This effective reduction of the ES impedance also depends on
the light intensity, which determines the rate of generation of
electron-hole pairs. In the absence of significant recombination,
the majority of photogenerated electrons flow out of the depletion
region and contribute to the photocurrent.
[0035] This light-induced surface-charge dependence may be used to
induce the lateral displacement of charged biomolecules between
fully exposed and partially masked regions of the interface. As the
illumination intensity is increased, the fully exposed regions will
correspond to the regions of interface of lowest impedance, and
hence of highest current, and biomolecules will be drawn into these
regions.
[0036] Additionally, time-varying changes in the illumination
pattern can be used to affect the motion of the biomolecule. For
example, charged biomolecules move in response to the change in
photopotential provided by the photon energy contacting the
depletion region of the semiconductor. If a focused beam of photon
energy is scanned to an adjacent spot on the photo-sensitive
semiconductive electrode, the biomolecule will move along with the
photon energy because the biomolecule will be attracted to this new
region having a change in current or photopotential. Where the
electrolyte medium comprises a sieve or gradient, this movement of
biomolecules results in the separation of the biomolecules based
upon size or other physical/chemical characteristics, a process
analogous to gel electrophoresis. By adjusting the speed at which
the photon energy beam moves to a different location on the
semiconductor surface, biomolecules can be separated with
precision. Thus, an electrolyte medium that is capable of
separating molecules based upon more than one physical/chemical
characteristic may be used wherein movement of a biomolecule in one
direction separates the biomolecule based upon one characteristic
while movement in a another direction (e.g., perpendicular to the
first) separates the biomolecules based upon a second
characteristic.
[0037] Turning now to FIG. 1, an apparatus, system, and method of
the invention utilize an electrolyte medium 400 (e.g., an ionically
conductive medium) sandwiched between a photo-sensitive
semiconductive electrode 300 and a counter electrode 200. A power
supply 600 (e.g., a potentiostat) is connected by wires 250 and 350
to the electrodes 200 and 300, respectively. The power supply 600
provides voltage to generate a potential difference between the
electrodes 200 and 300 and across the electrolyte medium 400. The
electrolyte medium 400 comprises analyte molecules to be analyzed
and/or separated.
[0038] The electrodes 200 and 300 and the electrolyte medium 400
form an electrochemical cell (generally depicted by 5 in FIGS. 2A
and B). The electrodes 200 and 300 are positioned relative to one
another such that when voltage is applied an electric field is
generated between the electrodes. In one aspect of the invention
the electrodes are planar and parallel and are separated from one
another to define a gap of about 20-50 .mu.m, about 50-100 .mu.m,
about 100 .mu.m to 1 mm, about 1-2 mm, or about 2-3 mm.
[0039] FIG. 2 shows an electrochemical cell of the invention in
more detail. The electrochemical cell includes a photo-sensitive
semiconductive electrode 300, an electrolyte medium 400, and a
counter electrode 200. In one aspect of the invention, an inlet
port 550 can be used to load the electrolyte medium 400 into the
space between the electrodes. The inlet port 550 may also be used
to load a sample to be analyzed.
[0040] The counter electrode 200 is typically semi-transparent
(e.g., phototransmissive). An example of a suitable
counter-electrode is indium tin oxide (ITO) deposited on a glass
substrate. The counter electrode may also include a silane layer
that prevents analyte biomolecules from adhering to the electrode.
Other transparent or semi-transparent materials can be used, such
as quartz with a thin layer of a conductive material, such as gold,
other inorganic conductors, or combinations of conductive
materials.
[0041] The photo-sensitive semiconductive electrode 300 can be
formed with a p-type or n-type material known in the art, such as
Si, Ge, GaAs, CdS, ZnO, TiO.sub.2, and the like (see Table 1).
Other semiconductive materials are discussed in the compendium
Semiconductor Electrodes, H. Finklea, ed., Elsevier, New York,
1988, which is incorporated herein by reference in its entirety.
One example of a photo-sensitive semiconductive electrode is an Si
electrode. In one aspect of the invention, the semiconductor (e.g.,
the Si) may also include an oxide layer (e.g., a SiOx layer). The
photo-sensitive semiconductive electrode can be fabricated from a 1
inch-square portion of a Si (100) wafer, typically 200-250 .mu.m
thick, n-doped to typically 0.01 Ohm cm resistivity. In one aspect
the Si (100) wafer may be capped with a thin oxide of typically
30-40 .ANG. in thickness. A thick oxide layer can be obtained by
growing under standard conditions in a furnace at 950.degree. C.,
which can be etched to obtain a desired structure. Alternatively, a
thin oxide layer may be regrown on a previously stripped surface of
(100)-orientation under UV light. UV oxide growth provides the
ability to pattern the surface by placing a quartz mask
representing the desired pattern in the path of the UV light and
thereby generating a chemically homogeneous topographically flat
surface. To avoid particle adsorption to the surface of the
photosensitive electrode, clean conditions should be used. For
example, the photo-sensitive semiconductive electrode is cleaned
prior to contacting an electrolyte medium to ensure that the
voltage potential can be applied across the photo-sensitive
semiconductive electrode and the electrolyte medium interface.
Oxides on a Ge substrate can be cathodically reduced or removed,
for example, using a mild etching solution.
1TABLE 1 Some Elemental and Compound Semiconductors For
Photoelectrochemical Application Conductive Optical Band Gap
Semiconductor Types(s) Energy [eV] Si n, p 1.11 GaAs n, p 1.42 GaP
n, p 2.26 InP n, p 1.35 CdS n 2.42 CdSe n 1.70 CdTe n, p 1.50
TiO.sub.2 n 3.00 (rutile) 3.20 (anatase) ZnO n 3.35
[0042] An ohmic contact is electrically connected to the
photo-sensitive semiconductive electrode 300 in order to maintain
the integrity of the potential applied across the photo-sensitive
semiconductive electrode/electrolyte medium interface. An ohmic
contact is typically a metal-semiconductor contact with a linear
current-voltage characteristic and low resistance. Such ohmic
contact materials are known in the art. Thus, as a result of the
low electrical resistance of the ohmic contact, the voltage applied
across the electrolyte medium 400 is dropped in the depletion
layer. Accordingly, the voltage applied can be less than 10 volts
or as high as 1 kV or more.
[0043] Also depicted in FIG. 1 is a photon energy source 50. The
photon energy source can be any type of light source that emits a
focused beam of photon energy 60 (e.g., light) or which may be
modified using various filters 110, mirrors 100, lens 150, and/or
apertures to direct a focused beam of photon energy 60. Filters
make it possible to adjust the luminous power to values of less
than, equal to, or greater than the necessary threshold. For
example, a helium-neon laser with output of 5 mW at 632.8 nm may be
used. Such a laser source provides about 500 .mu.W of power near a
target analyte biomolecule. In one aspect of the invention, the
beam of light may be modified to obtain a desired geometric shape
such as a focused point, a focused line of a millimeter or less in
length, a curved "parenthetical shaped" geometry, and the like.
Accordingly, photon energy sources include laser diodes. or light
emitting diodes that emit light in the visible or very
near-infrared wavelength ranges.
[0044] The photon energy source 50 will typically provide a focused
point of intense light so that the photon energy arriving at the
interface is highly localized. In one aspect of the invention, the
photon energy 60 is pulsed so that no saturation phenomenon occurs.
The magnitude of the photovoltage induced is related to the light
intensity and the extent of the band bending. The latter can be
controlled by adjusting the biasing voltage. In one embodiment, the
biasing is kept constant to maintain a depletion region in the
semiconductor. The bias is selected such that the photovoltage is
maximized. In one embodiment of the invention, a potential between
the photo-sensitive electrode 300 and counter electrode 200 can
also be alternated in such a way that the potential is alternated
between the electrodes (typically in the dark, "light off," phase).
This causes the analyte molecule (e.g., a biomolecule) motion to
alternate between the photo-sensitive electrode 300 and the counter
electrode 200, and thus inhibits the analyte molecules from
collecting on the surface of either of the electrodes.
[0045] In order to scan the photon energy source across the
photo-sensitive electrode 300 the beam of photon energy 60 or the
photo-sensitive electrode 300 are moved relative to one or the
other or both to each other. As depicted in FIG. 1 a Galvos 160 is
used to focus the photon energy 60 to various points on the
photo-sensitive electrode 300. The Galvos comprises automated means
164 and 168 for positioning the beam of photon energy 60 in an X
and Y plane. In another aspect of the invention, the Galvos and/or
the photo-sensitive semiconductive electrode may be located on a
Z-stage that further allows focusing of the beam of light in a
Z-plane. In yet another aspect of the invention, the
electrochemical cell comprising the photo-sensitive semiconductive
electrode is located on an automated X-Y stage or an automated
X-Y-Z stage. The automated stage allows for the automation of the
scanning of the beam of photon energy upon the photo-sensitive
semiconductive electrode. In another aspect, a pivotally mounted
mirror can be used to direct the photon energy (e.g., light beam)
across (e.g., at various coordinates of) the photo-sensitive
electrode. In another aspect of the invention an array of photon
energy beams can be used to move multiple molecules or samples
comprising molecules across the photo-sensitive electrode. Such an
array of photon energy beams can be generated by splitting one
photon energy beam into multiple beams or by utilizing multiple
photon energy sources.
[0046] The electrolyte medium 400 typically comprising, for
example, a TRIS buffer. In one embodiment, the electrolyte medium
further comprises a gel including, for example, polyacrylamide
(e.g., cross-linked polyacrylamide), agarose, dextran, and the
like, containing electrolyte and buffered substances, which are
brought into contact with the photo-sensitive semiconductive
electrode and the counter electrode. The electrolyte medium and/or
gel may be a few microns to a few millimeters thick (e.g., about
20-50 .mu.m, about 50-100 .mu.m, about 100 .mu.m to 1 mm, about 1-2
mm, or about 2-3 mm thick). The electrolyte medium may contain
analyte molecules (e.g., proteins, polypeptide, peptides,
polynucleotides, oligonucleotides, and the like) to be separated or
analyzed at the time of placing the electrolyte medium in contact
with the electrode(s). Alternatively, the analyte molecules may be
added to the electrolyte medium after the electrolyte medium is
contacted with the electrode(s). Techniques for forming "wells" or
sample reservoirs in various gel media such as agarose and
polyacrylamide for receiving analyte molecules are known in the
art. In addition, various physical characteristics of gel media
(e.g., agarose or polyacrylamide gels) can be modified to effect
the rate and movement of analyte molecules. For example, changing
the percentages of agarose or acrylamide in the electrolyte medium
will change the rate of movement and thus the type of separation
that can be accomplished between analyte molecules of different
sizes. In another aspect of the invention separation can be
accomplished by patterning the surface of the photo-sensitive
semiconductive electrode (See FIG. 4).
[0047] The apparatus, system and method thus includes an
electrolyte medium 400 sandwiched between the photo-sensitive
semiconductive electrode 300 and the counter electrode 200 to
complete an electrical circuit between the electrodes. When the
photo-sensitive semiconductive electrode is in contact with an
electrolyte medium 400, and a voltage is applied to the electrodes
such that a double layer of charge (dipole layer) is established at
the interface.
[0048] As shown in FIG. 3, when a voltage is applied to the
electrodes 200 and 300, a potential is generated between the
electrodes and across the electrolyte medium 400. The potential
bends the conduction and valence bands 36 and 38 in the
photo-sensitive electrode 300. Bending of the conduction band 36,
below the Fermi level 40 for the photo-sensitive electrode 300
creates a depletion region 18. The depletion region 18 can provide
a source of electrons or holes to create a photopotential in the
electrolyte medium 400.
[0049] With reference to FIG. 3A and B, the photon energy source
50, such as a laser, having an emission energy level greater than
the voltage potential across the photo-sensitive
electrode-electrolyte interface directs photon energy 60 at the
depletion region 18a or accumulation region 18b in the
photo-sensitive electrode 300. The photon energy source 50
produces, for example, a line (or point) of intense light across
the width of the depletion or accumulation region. As a result, the
electrons (when using a p-type semiconductive material; FIG. 3A)
from the generated electron-hole pairs are localized at the
interface. When using an n-type semiconductive material (FIG. 3B)
the holes are accumulated at the interface.
[0050] The photon energy source 50 raises the energy level of the
photo-sensitive electrode 300 at the interface with the electrolyte
medium 400 above the Fermi level to create electron-hole pairs in
the depletion region which are separated by the pre-existing
potential between the electrodes 200 and 300. The separated
electrons and holes have opposite charges, which cause the
electrons to move in a direction opposite of the holes. By proper
choice of the semiconductive material of the photo-sensitive
electrode (e.g., p-type or n-type) and counter electrode (anode or
cathode), either the electron or holes of the electron-hole pairs
can be brought to the interface with the electrolyte medium 400 to
generate a photopotential. For example, where a p-type material is
used, the electrons of the electron-hole pairs move to the
photo-sensitive electrode/electrolyte medium interface.
[0051] The direction of the photoelectrophoresis action is to the
irradiated spot on the semiconductor electrode. In one aspect of
the invention, a reversing potential is applied between the two
electrodes during a "light-off" cycle of the repetitive pulses.
This moves the molecules in the opposite direction so they can
again be attracted to the semiconductor when the focused laser beam
is moved to an adjacent spot. When a line of instantaneous
photopotential is created between the photo-sensitive
semiconductive electrode and counter electrode, the analyte
molecules move towards the counter electrode. Since the photon
energy can be incrementally stepped to a different location in the
depletion region of the photo-sensitive semiconductive electrode,
biomolecules are incrementally attracted to the current flux at the
different location and thereby are moved incrementally through the
electrolyte medium with an overall direction parallel to the two
electrodes. The biomolecules move in the same direction as the
incremental stepping of the photon energy beam. Interaction of the
biomolecules with a gel matrix of the electrolyte medium results in
the separation of molecules of varying sizes of, for example,
polymer length and/or molecular weight.
[0052] It may be desirable to prevent biomolecules present in an
electrolyte medium between two electrodes from contacting either
electrode. In order to avoid contact of the biomolecule in the
electrolyte medium with either electrode, applied voltage to the
electrodes may be intermittent or reversed at periodic intervals.
In yet another aspect, a combination of periodic or reversed
voltage and intermittent photon energy may be used.
[0053] FIG. 1 also shows a programmable computer 500. The various
techniques, methods, and aspects of the invention described above
can be controlled in part or in whole using computer-based systems
and methods. Additionally, computer-based systems and methods can
be used to augment or enhance the functionality described above,
increase the speed at which the functions can be performed, and
provide additional features and aspects as a part of or in addition
to those of the invention.
[0054] The programmable-computer system can include a main memory,
preferably random access memory (RAM), and can also include a
secondary memory. The secondary memory can include, for example, a
hard disk drive and/or a removable storage drive, representing a
floppy disk drive, a magnetic tape drive, an optical disk drive,
etc. The removable storage drive reads from and/or writes to a
removable storage medium. Removable storage media represents a
floppy disk magnetic tape, optical disk, etc., which is read by and
written to by removable storage drive. As will be appreciated, the
removable storage media includes a computer usable storage medium
having stored therein computer software and/or data.
[0055] In alternative embodiments, secondary memory may include
other similar means for allowing computer programs or other
instructions to be loaded into a computer system. Such means can
include, for example, a removable storage unit and an interface.
Examples of such can include a program cartridge and cartridge
interface (such as the found in video game devices), a movable
memory chip (such as an EPROM, or PROM) and associated socket, and
other removable storage units and interfaces which allow software
and data to be transferred from the removable storage unit to the
computer system.
[0056] The computer system can also include a communications
interface. Communications interfaces allow software and data to be
transferred between computer system and external devices. Examples
of communications interfaces can include a modem, a network
interface (such as, for example, an Ethernet card), a
communications port, a PCMCIA slot and card, etc. Software and data
transferred via a communications interface are in the form of
signals which can be electronic, electromagnetic, optical or other
signals capable of being received by a communications interface.
These signals are provided to communications interface via a
channel capable of carrying signals and can be implemented using a
wireless medium, wire or cable, fiber optics or other
communications medium. Some examples of a channel can include a
phone line, a cellular phone link, an RF link, a network interface,
and other communications channels.
[0057] Computer programs useful for monitoring or managing the
apparatus and systems of the invention are typically present on
computer program medium. A computer readable medium includes media
such as a removable storage device, a disk capable of installation
in a disk drive, and signals on a channel. These computer program
products are means for providing software or program instructions
to a computer systems.
[0058] Computer programs (also called computer control logic) are
stored in main memory and/or secondary memory. Computer programs
can also be received via a communications interface. Such computer
programs, when executed, enable the computer system to perform the
features of the present invention as discussed herein. In
particular, the computer programs, when executed, enable the
processor to perform the methods outlined in above. Accordingly,
such computer programs represent controllers of the system.
[0059] In an embodiment where the elements are implemented using
software, the software may be stored in, or transmitted via, a
computer program product and loaded into a computer system using a
removable storage drive, hard drive or communications interface.
The control logic (software), when executed by the processor,
causes the processor to perform the functions of the invention as
described herein.
[0060] In another embodiment, the elements are implemented
primarily in hardware using, for example, hardware components such
as PALs, application specific integrated circuits (ASICs) or other
hardware components. Implementation of a hardware state machine so
as to perform the functions described herein will be apparent to
person skilled in the relevant art(s). In yet another embodiment,
elements are implemented using a combination of both hardware and
software.
[0061] Transport of biomolecules has been demonstrated when
applying voltages less than 1 KV. Semiconductors such as Si, Ge,
GaAs, TiO.sub.2, CdS, and ZnO in contact with a liquid exhibit a
change in surface charge upon irradiation with light of an
appropriate wavelength when electronic bands of the semiconductors
are bent. These reactions occur initially by the absorption of
photons of energies greater than the corresponding semiconductor
band gap energy to form conduction band electron valence band hole
pairs
[0062] The following examples are provided to demonstrate a
particular aspect of the invention and should not be construed to
limit the invention.
EXAMPLES
[0063] A counter electrode was prepared by thermally evaporating
onto a glass slide (1 inches by 3 inches), 2.5 nm of chromium and
25 nm Gold on one surface of the slide. An AWG 26 wire was attached
to the chromium/gold using silver epoxy and connected to a Model
173 potentiostat/galvanostat (EG&G Princeton Applied
Research).
[0064] A photo-sensitive semiconductive electrode of Germanium
(n-type; <0.4 .OMEGA.-cm; 1-1-1 orientation; 2" .PHI.; 14 mil
thick) was obtained from Polishing Corp. of America
(part#Ge2N111SSP). An ohmic contact was provided using a gallium
indium eutectic 99.99% (Alfa Aesar #12478)--diamond scribe to
scratch the surface. Epoxy was used to connect an AWG 26 wire Lo
the electrode, which was in turn connected to a Model 173
potentiostat/galvanostat (EG&G Princeton Applied Research).
[0065] The counter electrode and semiconductive electrode were
arranged in a cell having an inner diameter of about 4 cm and a
height of about 6 mm. A Tris-glycine buffer (6.25 mM Trizma base,
62.5 glycine, pH 8.6) was used to cover a 1% (w/v) agarose gel. The
gel thickness is 1.85 mm. The gap between the two electrodes is
about 6 mm (e.g., 1.85 mm gel plus 4 mm Tris-buffer).
[0066] Bovine serum albumin, lysozyme, P-amylase, and/or rabbit IgG
were labeled with Marina Blue, an amine-reactive blue-fluorescent
dye having an absorbance at 362 nm and an emission at 459 nm.
(Molecular Probes, cat. # M-10165). A mineral lamp (366 nm) was
used to visualize the biomolecules (UVP Model UVGL-58).
[0067] Using current control the electrode current was set to
-0.235 mA (approx. 3.2V) on the potentiostat. The current was
cycled at various times. Visualization of the blue-labeled
biomolecule showed movement of the biomolecule in a verticle
zig-zag motion as the current of the potentiostat was cycled and as
the laser was scanned from one end of the gel to the other. FIG. 5,
shows a representation of the overall movement of a labeled bovine
serum albumin molecules as the laser was scanned and the current
cycled.
[0068] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
* * * * *